In 1994 cancer of the prostate gland is expected to be diagnosed in 200,000 men in the U.S. and 38,000 American males will die from this disease (Garnick, M. B. (1994). The Dilemmas of Prostate Cancer. Scientific American, April:72-81). Thus, prostate cancer is the most frequently diagnosed malignancy (other than that of the skin) in U.S. men and the second leading cause of cancer-related deaths (behind lung cancer) in that group.
Compositions useful in the treatment of prostatic cancer and related conditions are described in U.S. Pat. Nos. 5,599,686, 5,866,679 and 5,948,750, and U.S. patent applications Ser. No. 08/950,805, filed Oct. 14, 1997, now U.S. Pat. No. 8,948,750, (PCT Publ.No. WO 98/18493). Said compositions comprise chemical conjugates comprising known cytotoxic agents and oligopeptides having amino acid sequences that are selectively proteolytically cleaved by free prostate specific antigen and that include a cyclic amino acid having a hydrophilic substituent. The oligopeptide moieties are selected from oligomers that are selectively recognised by free prostate specific antigen (PSA) and are capable of being proteolytically cleaved by the enzymatic activity thereof.
Ideally, the cytotoxic activity of the cytotoxic agent is greatly reduced or absent when the intact oligopeptide containing the PSA proteolytic cleavage site is bonded directly, or through a chemical linker, to the cytotoxic agent. Also ideally, the cytotoxic activity of the cytotoxic agent increases significantly, or is restored completely, upon proteolytic cleavage of the attached oligopeptide at the cleavage site. Anthracycline antibiotics, in particular doxorubicin, are among the cytotoxic agents that were described in the published patent applications as preferably incorporated into such conjugates, which may be referred to as PSA conjugates.
It is the object of this invention to provide an efficient, scaleable and reproducible process for the preparation of PSA conjugates having an anthracycline antibiotic moiety as the cytotoxic agent and having the cleavable oligopeptide directly attached, via the C-terminus amino acid, to the glycosyl amine of the anthracycline antibiotic moiety.
Another object of this invention is to provide intermediate compounds useful in the preparation of such PSA conjugates.
A chemical process for the preparation of a PSA conjugate which comprises an anthracycline antibiotic and an oligopeptide, having an amino acid sequence that is selectively proteolytically cleaved by free prostate specific antigen (PSA) is disclosed. Such conjugates are useful in the treatment of prostatic cancer and benign prostatic hyperplasia (BPH).
The present invention is directed to a process for the preparation of compounds as illustrated by formula I: 
or a pharmaceutically acceptable salt thereof
xe2x80x83wherein
oligopeptide is an oligopeptide which is selectively recognized by the free prostate specific antigen (PSA) and is capable of being proteolytically cleaved by the enzymatic activity of the free prostate specific antigen,
Ra is xe2x80x94CH3, xe2x80x94CH2OH, xe2x80x94CH2OCO(CH2)3CH3, or xe2x80x94CH2OCOCH(OC2H5)2;
Rb is xe2x80x94OCH3, xe2x80x94OH or xe2x80x94H;
Rd is xe2x80x94OH, xe2x80x94OTHP or xe2x80x94H; and
Re is xe2x80x94OH or xe2x80x94H, provided that Re is not xe2x80x94OH when Rd is xe2x80x94OH or xe2x80x94OTHP;
R is selected from:
a) acetyl; 
e) hydrogen;
R1 and R2 are independently selected from:
a) hydrogen,
b) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, halogen, C1-C6 perfluoroalkyl, R3Oxe2x80x94, R3C(O)NR3xe2x80x94, (R3)2NC(O)xe2x80x94, R32Nxe2x80x94C(NR3)xe2x80x94, R4S(O)mNH, CN, NO2, R3C(O)xe2x80x94, N3, xe2x80x94N(R3)2, or R4C(O)NR3xe2x80x94,
c) unsubstituted C1-C6 alkyl,
d) substituted C1-C6 alkyl wherein the substituent on the substituted C1-C6 alkyl is selected from unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, R3Oxe2x80x94, R4S(O)mNH, R3C(O)NR3xe2x80x94, (R3)2NC(O)xe2x80x94, R32Nxe2x80x94C(NR3)xe2x80x94, CN, R3C(O)xe2x80x94, N3, xe2x80x94N(R3)2, and R4OC(O)xe2x80x94NR3xe2x80x94; or
R1 and R2 are combined to form xe2x80x94(CH2)sxe2x80x94 wherein one of the carbon atoms is optionally replaced by a moiety selected from: O, S(O)m, xe2x80x94NC(O)xe2x80x94, NH and xe2x80x94N(COR4)xe2x80x94;
R3 is selected from: hydrogen, aryl, substituted aryl, heterocycle, substituted heterocycle, C1-C6 alkyl and C3-C10 cycloalkyl;
R4 is selected from: aryl, substituted aryl, heterocycle, substituted heterocycle, C1-C6 alkyl and C3-C10 cycloalkyl;
m is 0, 1 or 2;
n is 1, 2, 3 or 4;
p is zero or an integer between 1 and 100;
q is 0 or 1, provided that if p is zero, q is 1;
r is an integer between 1 and 10; and
s is 3, 4 or 5.
The process comprises the step of preparing the compound of the formula Ia: 
or a salt thereof;
xe2x80x83wherein
oligopeptide, R, Ra, Rb, Rd and Re are described as above
Rxe2x80x2 is selected from:
a) R,
b) a protected precursor to R, and
c) an N-terminus protecting group;
by mixing an oligopeptide of the formula A: 
xe2x80x83wherein oligopeptide and Rxe2x80x2 are described as above, or a salt thereof;
with an anthracycline antibiotic of the formula B: 
xe2x80x83or a salt thereof,
wherein Ra, Rb, Rd and Re are as described hereinabove,
in the presence of a carboxyl activating agent and, optionally, in the presence of a base.
In an embodiment of the instant process, if Rxe2x80x2 is a protected precursor to R or an N-terminus protecting group, the process further comprises the step of removing the protecting group to produce the compound of the formula I.
In an embodiment of the instant process, the oligopeptide of formula A is mixed with a salt of the anthracycline antibiotic of the formula B in the presence of a carboxyl activating agent and a base.
In an embodiment of the instant process, the carboxyl activating agent is selected from dicyclohexylcarbodiimide (DCC), N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide (EDC), and 1,3-diisopropylcarbodiimide (DIC).
In a preferred embodiment, the carboxyl activating agent is EDC.
In an embodiment of the instant process, the base is selected from 2,4,6-collidine, lutidine, pyridine, triethyl amine and (iPr)2NEt.
In a preferred embodiment, the base is 2,4,6-collidine.
It has been surprisingly discovered that use 2,4,6-collidine as the base in the instant process for the formation of the compound of the formula Ia results in lower epimerization of the C-terminus amino acid moiety than when other bases previously described are utilized.
In a further embodiment of the instant process, the mixing of the compounds of formula A and formula B is additionally in the presence of an additive.
In a preferred further embodiment of the instant process the additive is selected from HOAt, HOBt, HOPO or a combination thereof.
In a preferred embodiment of the further embodiment of the instant process, the additive is HOAt.
In a second preferred embodiment of the further embodiment of the instant process, the additive is a combination of HOPO and HOAt.
In a further embodiment of the instant process, the oligopeptide of the formula A is mixed with the anthracycline antibiotic of the formula B in the presence of a carboxyl activating agent, an additive and a base.
In a preferred further embodiment of the instant process, the oligopeptide of the formula A is mixed with the anthracycline antibiotic of the formula B in the presence of an additive and a base, and a carboxyl activating agent is thereafter added to the mixture.
It has been surprisingly discovered that when the carboxyl activating agent is added last to a preformed mixture of the process components the amount of epimerization that occurs at the C-terminus amino acid of the oligopeptide moiety in the compound of formula Ia is less than when the base is added last to the reaction mixture.
In a more preferred further embodiment of the instant process, the oligopeptide of the formula A is mixed with the anthracycline antibiotic of the formula B in the presence of an additive and a base, and a carboxyl activating agent is thereafter added to the mixture in two or more portions.
In an embodiment of the instant invention, the anthracycline antibiotic is doxorubicin (Ra is xe2x80x94CH2OH, Rb is xe2x80x94OCH3, Rc is H and Rd is xe2x80x94OH).
In another embodiment of the instant invention, R is selected from:
a) acetyl; and
b) 
In another embodiment of the instant invention, R is 
and Rxe2x80x2 is 
In another embodiment of the instant invention, R is 
and Rxe2x80x2 is 
and the process further comprises the step of removing the protecting group by contacting the compound of formula Ia with piperidine.
In a preferred embodiment of the instant invention, the oligopeptide comprises an oligomer selected from:
a) 4-HypXaaSerTyrGln|SerSer (SEQ.ID.NO.: 1),
b) 4-HypXaaSerTyrGln|SerAla (SEQ.ID.NO.: 2),
c) Ala-4-HypXaaSerTyrTyr|Ser (SEQ.ID.NO.: 3),
d) AlaAsn4-HypXaaSerTyrGln|Ser (SEQ.ID.NO.: 4),
e) 4-HypXaaSerTyrGln|SerSerThr (SEQ.ID.NO.: 5),
f) 4-HypTyrGln|SerSerThr (SEQ.ID.NO.: 6),
g) 4-HypXaaSerTyrGln|SerSerSer (SEQ.ID.NO.: 7),
h) 4-HypTyrGln|SerSerSer (SEQ.ID.NO.: 8),
i) 4-HypXaaLysTyrGln|SerSerSer (SEQ.ID.NO.: 9),
j) 4-HypXaahArgTyrGln|SerSerSer (SEQ.ID.NO.: 10),
k) 4-HypXaaSerTyrGln|SerSerLeu (SEQ.ID.NO.: 11);
l) 4-HypTyrGln|SerSerLeu (SEQ.ID.NO.: 12);
m) 4-HypXaaSerTyrGln|SerLeu (SEQ.ID.NO.: 13);
n) 4-HypTyrGln|SerLeu (SEQ.ID.NO.: 14);
p) 4-HypXaaSerTyrGln|SerNle (SEQ.ID.NO.: 15);
q) 4-HypTyrGln|SerNle (SEQ.ID.NO.: 16);
r) 4-HypXaaSerTyrGln|SerTIC (SEQ.ID.NO.: 17);
s) 4-HypTyrGln|SerTIC (SEQ.ID.NO.: 18);
t) 4-HypXaaSerChgGln|SerLeu (SEQ.ID.NO.: 19);
u) 4-Hyp ChgGln|SerLeu (SEQ(ID.NO.: 20);
v) 4-HypXaaSerChgGln|SerNle (SEQ.ID.NO.: 21);
w) 4-HypChgGln|SerNle (SEQ.ID.NO.: 22);
x) 4-HypXaaSerChgGln|SerTIC (SEQ.ID.NO.: 23);
y) 4-HypChgGln|SerTIC (SEQ.ID.NO.: 24);
z) AlaSerTyrGln|SerSerLeu (SEQ.ID.NO.: 25);
aa) SerhArgChgGln|SerLeu (SEQ.ID.NO.: 26);
bb) hArgSerSerTyrGln|SerNle (SEQ.ID.NO.: 27);
cc) hArgAlaSerChgGln|SerLeu (SEQ.ID.NO.: 28);
dd) hArgSerSerTyrGln|SerLeu (SEQ.ID.NO.: 29);
ee) hArgSerSerChg|SerLeu (SEQ.ID.NO.: 30);
ff) SerhArgChgGln|SerLeu (SEQ.ID.NO.: 31);
gg) hArgTyrGln|SerLeu (SEQ.ID.NO.: 32);
hh) hArgSerSerChgGln|SerLeu (SEQ.ID.NO.: 33);
ii) SerhArgTyrGln|SerLeu (SEQ.ID.NO.: 34);
jj) SerSerTyrGln|SerLeu (SEQ.ID.NO.: 35);
kk) SerSerSerChgGln|SerLeu (SEQ.ID.NO.: 36);
ll) 3PAL-SerSerChgGln|SerLeu (SEQ.ID.NO.: 37);
mm) SerSerChgGln|SerLeu (SEQ.ID.NO.: 38);
nn) SerSerSerChgGln|Ser(dLeu) (SEQ.ID.NO.: 39);
oo) SerSerSerChgGln|SerVal (SEQ.ID.NO.: 40);
pp) ProSerSerChgGln|SerVal (SEQ.ID.NO.: 41);
qq) GlySerSerChgGln|SerLeu (SEQ.ID.NO.: 42);
rr) hSerSerSerChgGln|SerLeu (SEQ.ID.NO.: 43);
ss) hArgSerSerChgGln|SerNle (SEQ.ID.NO.: 44);
tt) hArgTyrGln|SerSerSerLeu (SEQ.ID.NO.: 45);
uu) LysTyrGln|SerSerSerLeu (SEQ.ID.NO.: 46);
vv) SerTyrGln|SerSerSerLeu (SEQ.ID.NO.: 47);
ww) SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 48);
xx) 3PAL-SerSerChgGln-Ser(dLeu) (SEQ.ID.NO.: 49); and
yy) AlaSerChgGln-SerLeu (SEQ.ID.NO.: 50).
xe2x80x83wherein 4-Hyp is 4-hydroxyproline, Xaa is any amino acid, hArg is homoarginine, hSer is homoserine, TIC is 1,2,3,4-tetrahydro-3-isoquinoline carboxylic acid, Cha is cyclohexylalanine and Chg is cyclohexylglycine.
Preferably Xaa in the more preferred embodiment is selected from Ala, Ser and Ile.
In a preferred embodiment the present invention is directed to a process for the preparation of the compound of formula 4: 
or a pharmaceutically acceptable salt thereof;
which comprises the step of mixing an oligopeptide of the formula 2a: 
xe2x80x83wherein Rxe2x80x3 is xe2x80x94OH or a protected precursor to xe2x80x94OH;
or a salt thereof;
with an anthracycline antibiotic of the formula 1: 
xe2x80x83or a salt thereof;
in the presence of a carboxyl activating agent and, optionally, in the presence of a base, to form a compound of the formula 3a: 
xe2x80x83or a salt thereof.
In a further embodiment of this preferred embodiment, the process further comprises the step of converting Rxe2x80x3 to a xe2x80x94OH moiety.
In a more preferred embodiment, the present invention is directed to a process for the preparation of the compound of formula 4: 
or a pharmaceutically acceptable salt thereof;
which comprises the steps of:
a) mixing an oligopeptide of the formula 2: 
xe2x80x83or a salt thereof;
with an anthracycline antibiotic of the formula 1: 
xe2x80x83or a salt thereof;
in the presence of a carboxyl activating agent and, optionally, in the presence of a base, to form a compound of the formula 3: 
xe2x80x83or a salt thereof.
In a further embodiment of the more preferred embodiment of the instant process, the oligopeptide of the formula 2 is added to the reaction mixture that comprises a solvent, in particular DMF, at about xe2x88x926xc2x0 C. to about xe2x88x923xc2x0 C. to form a first slurry and the anthracycline antibiotic of the formula 1 is added to that slurry to form a second slurry. It has been surprisingly discovered that maintaining such a low temperature for addition of the oligopeptide to the reaction mixture and then adding the doxorubicin to the slurry at low temperature avoids the formation of a gel in the reaction mixture which is detrimental to a large scale preparation of the compound of the formula 3.
In a second further embodiment of the more preferred embodiment of the instant process, the anthracycline antibiotic of the formula 1 is mixed in a solvent, in particular DMF, optionally with an additive and optionally with a base, to form a first slurry, which is then cooled to about xe2x88x926xc2x0 C. to about xe2x88x923xc2x0 C. and the oligopeptide of the formula 2 is added to that slurry to form a second slurry.
In an embodiment of the further embodiments of the instant process, the oligopeptide of formula 1 is mixed with a salt of the anthracycline antibiotic of the formula 2 in the presence of a carboxyl activating agent and a base. Preferably, the salt of the anthracycline antibiotic of the formula 2 is the hydrochloride salt.
In an embodiment of the further embodiments of the instant process, the process further comprises the step of removing the fluorenylmethoxy protecting group of the compound of formula 3 to produce the compound of the formula 4.
In a further embodiment of the instant process, the step of removing the fluorenylmethoxy protecting group of the compound of formula 3 comprises contacting the compound of formula 3 with piperidine.
The phrase xe2x80x9coligomers that comprise an amino acid sequencexe2x80x9d as used hereinabove, and elsewhere in the Detailed Description of the Invention, describes oligomers of from about 3 to about 100 amino acids residues which include in their amino acid sequence the specific amino acid sequence decribed and which are therefore proteolytically cleaved within the amino acid sequence described by free PSA. Preferably, the oligomer is from 5 to 10 amino acid residues. Thus, for example, the following oligomer:
hArgSer4-HypChgGln|SerLeu (SEQ.ID.NO.: 53); comprises the amino acid sequence:
4-HypChgGln|SerLeu (SEQ.ID.NO.: 54); and would therefore come within the instant invention.
The inclusion of the symbol xe2x80x9c|xe2x80x9d within an amino acid sequence indicates the point within that sequence where the oligopeptide is proteolytically cleaved by free PSA.
The compounds of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention. Unless otherwise specified, named amino acids are understood to have the natural xe2x80x9cLxe2x80x9d stereoconfiguration. When any variable (e.g. aryl, heterocycle, R3 etc.) occurs more than one time in any constituent, its definition on each occurence is independent of every other occurence. For example, HO(CR1R2)2xe2x80x94 represents HOCH2CH2xe2x80x94, HOCH2CH(OH)xe2x80x94, HOCH(CH3)CH(OH)xe2x80x94, etc. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
The following abbreviations are utilized in the specification and tables to denote the indicated amino acids and moieties:
As used herein, xe2x80x9calkylxe2x80x9d and the alkyl portion of aralkyl and similar terms, is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; xe2x80x9calkoxyxe2x80x9d represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge.
As used herein, xe2x80x9ccycloalkylxe2x80x9d is intended to include non-aromatic cyclic hydrocarbon groups having the specified number of carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
xe2x80x9cAlkenylxe2x80x9d groups include those groups having the specified number of carbon atoms and having one or several double bonds. Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, isoprenyl, famesyl, geranyl, geranylgeranyl and the like.
xe2x80x9cAlkynylxe2x80x9d groups include those groups having the specified number of carbon atoms and having one triple bonds. Examples of alkynyl groups include acetylene, 2-butynyl, 2-pentynyl, 3-pentynyl and the like.
xe2x80x9cHalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as used herein means fluoro, chloro, bromo and iodo.
As used herein, xe2x80x9caryl,xe2x80x9d and the aryl portion of aralkyl and aroyl, is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
The term heterocycle or heterocyclic, as used herein, represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyt, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl, 2-oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, and thienyl.
As used herein in the terms xe2x80x9csubstituted C1-8 alkylxe2x80x9d, xe2x80x9csubstituted arylxe2x80x9d and xe2x80x9csubstituted heterocyclexe2x80x9d include moieties containing from 1 to 3 substituents in addition to the point of attachment to the rest of the compound. Such additional substituents are selected from F, Cl, Br, CF3, NH2, N(C1-C6 alkyl)2, NO2, CN, (C1-C6 alkyl)Oxe2x80x94, xe2x80x94OH, (C1-C6 alkyl)S(O)mxe2x80x94, (C1-C6 alkyl)C(O)NHxe2x80x94, H2Nxe2x80x94C(NH)xe2x80x94, (C1-C6 alkyl)C(O)xe2x80x94, (C1-C6 alkyl)OC(O)xe2x80x94, N3, (C1-C6 alkyl)OC(O)NHxe2x80x94 and C1-C20 alkyl.
The term xe2x80x9can integer between 1 and 10xe2x80x9d represents the numbers 1 and 10 as well as those integers between those numbers. The term xe2x80x9can integer between 1 and 100xe2x80x9d represents the numbers 1 and 100 as well as those integers between those numbers.
When R1 and R2 are combined to form xe2x80x94(CH2)sxe2x80x94, the cyclic moieties and heteroatom-containing cyclic moieties so defined include, but are not limited to: 
As used herein, the term xe2x80x9cPEGxe2x80x9d represents certain polyethylene glycol containing substituents having the designated number of ethyleneoxy subunits. Thus the term PEG(2) represents 
and the term PEG(6) represents 
As used herein, the term xe2x80x9c(2R)(2,3-dihydroxypropionyl)xe2x80x9d represents the following structure: 
As used herein, the term xe2x80x9c(2R,3S) 2,3,4-trihydroxybutanoylxe2x80x9d represents the following structure: 
The following compounds are specific examples of a oligopeptide-cytotoxic agent conjugate that may be prepared by the process of the instant invention: 
wherein X is: 
or an optical isomer or pharmaceutically acceptable salt thereof.
Other PSA conjugate compounds that may be prepared by the process of the instant invention include, but are not limited to, the compounds described in the following patents and patent publications:
U.S. Pat. No. 5,599,686, issued Feb. 4, 1997;
U.S. Pat. No. 5,866,679, issued Feb. 2, 1999;
WO 96/00503 (Jan. 11, 1996); U.S. Ser. No. 08/468,161 filed on Jun. 6, 1997 now U.S. Pat. No. 6,143,864;
WO 98/10651 (Mar. 19, 1998); U.S. Ser. No. 08/926,412 filed on Sep. 9, 1997 now U.S. Pat. No. 5,978,362; and
U.S. Pat. No. 5,948,750, issued Sep. 7, 1999; WO 98/18493 (May 7, 1998);
U.S. Ser. No. 08/950,805, filed on Oct. 14, 1997, now U.S. Pat. No. 5,948,750.
The pharmaceutically acceptable salts of the conjugates that may be prepared by the process of this invention and salts of Compounds of the formulae A, B, 1, 2a and 3a utilized in the instant processes include the conventional non-toxic salts of the compounds, e.g., non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.
With respect to the conjugates described herein which comprise a N-terminus blocking group on the peptide which contains a carboxylic acid moiety, a pharmaceutically acceptable salt may take the form xe2x80x94COOM, where M is a negative charge, which is balanced by a counterion, e.g., an alkali metal cation such as sodium or potassium. Other pharmaceutically acceptable counterions may be calcium, magnesium, zinc, ammonium, or alkylammonium cations such as tetramethylammonium, tetrabutylammonium, choline, triethylhydroammonium, meglumine, triethanolhydroammonium and the like.
For the purpose of the process of the instant invention, the carboxyl activating agent may be selected from the group including, but not limited to, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (known as HBTU), 1-hydroxybenzotriazole hydrate (known as HOBt), dicyclohexylcarbodiimide (DCC), N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide (EDC), diphenylphosphorylazide (DPPA), benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP), 1,3-diisopropylcarbodiimide (DIC) and the like, used in combination or singularly. Preferably the carboxyl activating agent is selected from EDC, DIC and DCC. Most preferably the carboxyl activating agent is EDC.
The coupling reaction of the amine moiety to the carboxyl terminus may also comprise a base, such as 2,4,6-collidine, lutidine, pyridine, triethyl amine, Hxc3xcnig""s base ((iPr)2NEt) and the like. Preferably the base is selected from 2,4,6-collidine and lutidine. Most preferably the base is 2,4,6-collidine. The coupling reaction may also comprise an additive, such as 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), 2-hydroxypyridine-N-oxide, (HOPO), N-hydroxysuccinimide, pyridine N-oxide and the like, or combinations thereof Preferably the additive is selected from HOAt, HOPO, HOBt or combinations thereof. More preferably the additive is HOAt. Also, more preferable the additive is a combination of HOPO and HOAt. The coupling reaction may also comprise a solvent. Such a solvent may be selected from N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc), N-methylpiperidone (NMP), aqueous THF, and the like. Preferably the solvent is selected from a polar aprotic organic solvent, such as DMF, DMAc, NMP and the like. Most preferably, the solvent is DMF.
One skilled in the art understands that in the synthesis of compounds of the invention, one may need to protect various reactive functionalities on the starting compounds and intermediates while a desired reaction is carried out on other portions of the molecule. After the desired reactions are complete, or at any desired time, normally such protecting groups will be removed by, for example, hydrolytic or hydrogenolytic means. Such protection and deprotection steps are conventional in organic chemistry. One skilled in the art is referred to Protective Groups in Organic Chemistry, McOmie, ed., Plenum Press, N.Y., N.Y. (1973); and, Protective Groups in Organic Synthesis, Greene, ed., John Wiley and Sons, N.Y., N.Y. (1981) for the teaching of protective groups which may be useful in the preparation of compounds of the present invention.
By way of example only, useful amino-protecting groups may include, for example, C1-C10 alkanoyl groups such as formyl, acetyl, dichloroacetyl, propionyl, hexanoyl, 3,3-diethylhexanoyl, xcex3-chlorobutryl, and the like; C1-C10 alkoxycarbonyl and C5-C15 aryloxycarbonyl groups such as tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl, 4-nitrobenzyloxycarbonyl, fluorenylmethyloxycarbonyl and cinnamoyloxycarbonyl; halo-(C1-C10) alkoxycarbonyl such as 2,2,2-trichloroethoxycarbonyl; and C1-C15 arylalkyl and alkenyl group such as benzyl, phenethyl, allyl, trityl, and the like. Other commonly used amino-protecting groups are those in the form of enamines prepared with xcex2-keto-esters such as methyl or ethyl acetoacetate.
Useful carboxy-protecting groups may include, for example, C1-C10 alkyl groups such as methyl, tert-butyl, decyl; halo-C1-C10 alkyl such as 2,2,2-trichloroethyl, and 2-iodoethyl; C5-C15 arylalkyl such as benzyl, 4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenylmethyl; C1-C10 alkanoyloxymethyl such as acetoxymethyl, propionoxymethyl and the like; and groups such as phenacyl, 4-halophenacyl, allyl, dimethylallyl, tri-(C1-C3 alkyl)silyl, such as trimethylsilyl, xcex2-p-toluenesulfonylethyl, xcex2-p-nitrophenylthio-ethyl, 2,4,6-trimethylbenzyl, xcex2-methylthioethyl, phthalimidomethyl, 2,4-dinitro-phenylsulphenyl, 2-nitrobenzhydryl and related groups.
Similarly, useful hydroxy protecting groups may include, for example, the formyl group, the chloroacetyl group, the benzyl group, the benzhydryl group, the trityl group, the 4-nitrobenzyl group, the trimethylsilyl group, the phenacyl group, the tert-butyl group, the methoxymethyl group, the tetrahydropyranyl group, and the like.
With respect to the preferred embodiment of an oligopeptide combined with the anthracycline antibiotic doxorubicin, the following Reaction Schemes illustrate the synthesis of the conjugates of the instant invention. Other bases than piperidine are useful in the deprotection illustrated in Reaction Scheme IV, including dimethyl amine, diethyl, amine, pyrrolidine, quinuclidine and the like. Preferably piperidine is utilized. 
Reaction Schemes V-VIII below illustrate other methods for the preparation of the PSA conjugates, in particular, the preparation of the compound of formula 4. Thus, as shown in Reaction Scheme V, an unblocked glutaryl peptide 5 may be prepared by methods well known in the art and coupled to the amine moiety of the anthracycline cytotoxic agent as described above. Specific preparation of the compound of formula 4 is illustrated in Reaction Scheme VI.
Alternatively, as illustrated in Reaction Scheme VII, a bis-blocked peptide 6 may be prepared by methods well known in the art and selectively deprotected to provide the benzyl ester analog of the compound of formula 2. After coupling to the anthracycline cytotoxic agent as described above, deprotection of the acid moiety can be accomplished by catalytic hydrogenation, thereby avoiding prolonged exposure of compound 4 to a base. Other carboxylic acid protecting groups that may be useful include t-butyl, sillyl and the like. It is known that the anthracyclines are chemically sensitive to base.
In yet another embodiment, the cleavable peptide portion of the instant conjugate may be attached in two or more fragments, as ilustrated in Reaction Scheme VIII. Such a synthetic strategy might allow more flexibility in selection and use of blocking groups. The particular selection of which peptide fragment to attach first that is shown in the scheme is illustrative only and is not meant to limiting. 